Project Summary/Abstract Human enteric pathogens are responsible for millions of deaths worldwide each year, and unfortunately, the current human cell culture models that exist are poorly suited to study many diseases. Therefore, the overarching goal of this U19 proposal is to implement the use of Human Intestinal Organoids (HIOs) as a novel model system to study enteric pathogens. HIOs are complex, multicellular 3-dimensional intestinal tissues generated from human pluripo- tent stem cells. In order to implement HIOs as a novel model for the investigation of enteric pathogenesis, we will investigate the use of this model to study HIO-pathogen interactions (Project 1), HIO-immune interactions (Project 2), and finally, we will engineer scalable fluidics systems that will also allow us to create luminal flow, take real-time physiological measurements and maintain HIOs in long-term culture (Project 3). Central to all 3 projects are the use of HIOs, and essential for Project 2 and Project 3 are generating genetically modified HIOs, and co-culture of im- mune cells with HIOs. While growth, differentiation and manipulation of hPSCs to generate HIOs is a well- characterized and robust process, centralization of these procedures will allow the most efficient use of project re- sources and will ensure uniform and standardized starting material for all projects. Therefore, the Stem Cell Differ- entiation Core (SCDC) will support all three U19 projects by carrying out the following four essential functions: 1) Provide centralized HIO production and quality control; 2) Provide quality controlled reagents, training and consultation required to differentiate hPSCs and grow HIOs; 3) Produce isogenically matched NOD2 and ATG16L1 genetically modified hPSC lines. 4) Differentiate macrophages, dendritic cells and neutrophils cells from hPSCs.